WO2019034030A1 - Identification method and content measurement method for pediatric compound endothelium corneum gigeriae galli chewable tablet - Google Patents

Abstract

The present invention relates to a quality control method for Chinese patent drugs, and specifically relates to an identification method and a content measurement method for a pediatric compound endothelium corneum gigeriae galli chewable tablet. Identification is carried out by using specific thin-layer chromatographic identification conditions, and a content is measured by using specific high-performance liquid chromatographic conditions, accordingly, the specificity and the accuracy are strong, chemical components of the product can be fully reflected, the sensitivity is high, the reproducibility is high, the operations are simple, and the change condition of the product quality can be reflected more objectively, comprehensively and sensitively, so that the product quality is controlled on the whole, and comprehensive monitoring of the quality of traditional Chinese drugs is implemented.

Description

Identification and content determination method of pediatric compound chicken inner chewable tablets Technical field

The invention relates to a traditional Chinese medicine quality control method, in particular to a method for identifying and contenting a chicken compound chicken chewable tablet.

Background technique

In the prior art, the pediatric compound chicken inner gold chewable tablet is included in the national registration standard [YBZ03852008], and is a Chinese medicine product exclusively produced by Henan Tailong Pharmaceutical Co., Ltd. Prescription: 85g of chicken gold, 165g of Liushen. Method: The above two flavors, crushed into fine powder, sifted, added auxiliary materials, mixed, made into granules, dried, pressed into 1000 pieces, that is. Traits: This product is a colored chewable tablet; it is fragrant and sweet. The prescription Chinese herbal medicines are chicken inner gold and Liushenqu, in which the chicken inner gold is the inner wall of the dry sandbag of the animal chicken. Liushenqu is a koji agent made from the co-fermentation of Spicy Scallop, Artemisia annua, Xanthium, Chixiaodou, Bitter Almond, Wheat Bran and Flour. The main ingredients are rutin, artemisinin, Xanthium, ferulic acid and so on.

At present, the existing quality testing of pediatric compound chicken inner chewable tablets is mainly based on the following criteria:

(1) Identification: Take the fine powder under the measurement of the content of the chicken chewable tablets in the compound chicken. Observe under the microscope: the stone cells are orange-yellow, shell-shaped, the wall is thicker, and the width of one side is obvious.

(2) Determination of content: Take 20 tablets of chicken compound chewable tablets, accurately weighed, finely ground, take about 2g of powder, accurately weighed, according to the first method of Chinese Pharmacopoeia nitrogen determination method, according to Chinese Pharmacopoeia nitrogen determination The second method of the method is carried out. Each tablet contains no less than 12.0 mg of nitrogen.

(3) Heavy metal inspection: Take about 1.0g of the test sample and inspect it according to the method of the second method of the Chinese Pharmacopoeia heavy metal inspection method. Heavy metals should not exceed 20 parts per million.

(4) Others shall comply with the relevant provisions of the Chinese Pharmacopoeia tablets.

The above-mentioned quality standards have the disadvantages that the identification is too simple, the microscopic identification is only the quality control index of Liuququ, and there is no control of the chicken internal gold; the determination of nitrogen element is the control of large-scale components, the specificity and repeatability are poor, and it cannot be quantitatively comprehensive. Test its active ingredients.

At present, there is no project involving thin layer identification and component quantitative testing in the current national standards for two-flavored herbs.

Summary of the invention

The object of the present invention is to provide a method for identifying and contenting a compound chicken inner chewable tablet with strong specificity, high sensitivity, good repeatability and reasonable quality control, which can comprehensively control the quality standard of the product.

The invention provides a method for determining and contenting a pediatric compound chicken inner chewable tablet, characterized in that the method comprises:

(1) a step of identifying by thin layer chromatography, wherein

The conditions of the thin layer chromatography are as follows: a thin layer chromatography plate of silica gel G is used as a thin layer chromatography plate, and the lower layer is placed under chloroform-ethyl acetate-methanol-water (12:3:8:1) at 10 ° C or lower. Liquid is a developing agent;

(2) a step of performing content determination by high performance liquid chromatography, wherein

The high performance liquid chromatography conditions are as follows: octadecylsilane bonded silica as a stationary phase, methanol-water-glacial acetic acid in a volume ratio (85:15:0.45) as mobile phase A, and acetonitrile-isopropyl The alcohol-water was mobile phase B in a volume ratio (50:20:30), and the gradient elution was carried out according to the following conditions:

Time (minutes)	Mobile phase A (%)	Mobile phase B (%)
0	0	100
12	0	100
20	30	70
32	85	15
34	15	85
46	15	85

Preferably, the step of identifying according to step (1) further comprises the step of using a macroporous adsorption resin column chromatography on the pediatric compound chicken inner gold chewable tablet.

Preferably, the step of macroporous adsorption resin column chromatography comprises: mixing the pediatric compound chicken inner gold chewable tablet with a mass fraction of 75% methanol solution by weight ratio of 4:25, sonicating, and then using macroporous adsorption resin. The column is preferably subjected to chromatography using an AB-8 macroporous adsorption resin column, and after elution adsorption equilibrium, the macroporous adsorption resin column is eluted with an ethanol solution having a mass fraction of 70% as an eluent, and the eluate is collected after the eluate is collected. Evaporate to dryness and add analytically pure methanol solution.

Preferably, the step of identifying the step (1) uses a chicken-gold-methanol solution as a reference drug solution to

The acid-rutin-methanol solution was used as a reference solution.

Preferably, the step of identifying by the thin layer chromatography in the step (1) comprises:

(1) preparing a sample solution for the pediatric compound chicken inner gold chewable tablet according to the step of macroporous adsorption resin column chromatography;

(2) preparing a chicken gold-methanol reference drug solution according to the step of macroporous adsorption resin column chromatography on the chicken internal gold reference material;

(3) taking ferulic acid reference substance and rutin reference substance, adding methanol to obtain ferulic acid-rutin-methanol reference solution;

(4) The above three solutions were aspirated and identified according to the conditions of the thin layer chromatography.

Preferably, the step of identifying by the thin layer chromatography in the step (1) comprises:

(1) The pediatric compound chicken inner chewable tablets were pulverized, mixed with a mass fraction of 75% methanol solution at a weight-to-volume ratio of 4:25, sonicated for 30 min, filtered, and the upper AB-8 macroporous adsorption resin column layer Analyze, wash with water until the washing liquid is nearly colorless, then elute with 35% ethanol and 70% ethanol in sequence, collect 70% ethanol eluent, evaporate dry, add methanol to dissolve, then get the test solution ;

(2) taking chicken internal gold reference medicine, according to the method of step (1) to obtain a chicken gold-methanol reference medicine solution;

(3) taking ferulic acid reference substance and rutin reference substance, adding methanol to make a mixed solution containing 0.5 mg of ferulic acid and 0.5 mg of rutin per 1 ml as a reference solution;

(4) Aspirate the above three solutions, respectively, on a high-performance silica gel G thin-layer plate, and use chloroform-ethyl acetate-methanol-water to develop a volume ratio of 12:3:8:1 as a developing agent, and take it out to dry. Spray with a mass fraction of 10% sulfuric acid in ethanol, heat at 105 ° C until the spots are clear, and examine them under sunlight and ultraviolet light (365 nm).

Preferably, the content determining step according to the step (2) further comprises the step of purifying the pediatric compound chicken inner gold chewable tablet by using a neutral alumina column.

Preferably, the step of purifying the neutral alumina column comprises: mixing the pediatric compound chicken inner gold chewable tablet with the chromatographic pure methanol according to a weight ratio of 1:2.5, sonicating, and purifying through a neutral alumina column to The neutral ethanol column was eluted with an absolute ethanol solution as an eluent, and the eluent was collected and evaporated to dryness, and purified by chromatography.

Preferably, the content determination step described in the step (2) uses an artemisinin-p-hydroxybenzaldehyde-Xan Erting-methanol solution as a reference solution.

Preferably, the step of determining the content by the high performance liquid chromatography in the step (2) comprises:

(1) preparing a test solution by purifying the pediatric compound chicken inner gold chewable tablet according to the neutral alumina column;

(2) taking artemisinin, p-hydroxybenzaldehyde and Xan Erting reference substance, adding methanol to make a mixed solution of artemisinin-p-hydroxybenzaldehyde-Cangling-methanol as a reference solution;

(3) Aspirating the above two solutions, injecting into a high performance liquid chromatograph, and performing content determination according to the high performance liquid chromatography conditions.

Preferably, the step of determining the content by the high performance liquid chromatography in the step (2) comprises:

(1) The pediatric compound chicken inner chewable tablets were pulverized, mixed with the chromatographic pure methanol solution at a weight-to-volume ratio of 1:2.5, sonicated for 30 minutes, allowed to cool, filtered, and passed through a neutral alumina column (100~). Purification by 120 mesh), elution with absolute ethanol, collecting the eluent, concentrating under reduced pressure, dissolving in chromatographically pure methanol, and filtering to obtain a test solution;

(2) Take artemisinin, p-hydroxybenzaldehyde and Xan Erting reference substance, add methanol to make a mixed solution containing 1mg of artemisinin, 0.2mg of p-hydroxybenzaldehyde and 0.1mg of Xanthing. Solution

(3) Aspirating the above two solutions and injecting them into a high performance liquid chromatograph, the high performance liquid chromatography conditions are: using octadecylsilane bonded silica as a stationary phase, the column temperature of the column is 38 ° C, with methanol - Water-glacial acetic acid was mobile phase A in a volume ratio (85:15:0.45), and mobile phase B in a volume ratio (50:20:30) in acetonitrile-isopropanol-water. Gradient elution was carried out according to the following conditions. The flow rate of the mobile phase is 1 ml / min;

Time (minutes)	Mobile phase A (%)	Mobile phase B (%)
0	0	100
12	0	100
20	30	70
32	85	15
34	15	85
46	15	85

The effect of the invention:

The specific thin layer chromatography conditions of the present invention are used for identification, and specific high performance liquid chromatography conditions are used for content determination, and the specificity and accuracy are strong, which can fully reflect the chemical composition of the product, high sensitivity, good reproducibility, and operation. Simple, more objective, comprehensive and sensitive reflects the changes in product quality, thus overall control of drug quality, to achieve comprehensive monitoring of the quality of traditional Chinese medicine.

DRAWINGS

Figure 1 is a linear view of the artemisinin reference substance;

Figure 2 is a linear view of the p-hydroxybenzaldehyde reference product;

Figure 3 is a linear view of the Xan Erting reference.

Detailed ways

The invention will now be described in detail by way of examples. It is to be understood that the present invention is not intended to be construed as limiting the invention.

Example 1

1. Pediatric compound chicken inner chewing tablets thin layer identification method

1.1 Instruments and reagents

Reagents: methanol, ethanol, chloroform, ethyl acetate, sulfuric acid are analytically pure, AB-8 macroporous adsorption resin column, silica gel G thin layer plate.

Sample to be tested - Pediatric compound chicken inner chewing tablets (batch number: 1610222; 1.2g / piece): Henan Tailong Pharmaceutical Co., Ltd. provided.

Negative sample 1 (excluding chicken gold): provided by Henan Tailong Pharmaceutical Co., Ltd.

Negative sample 2 (excluding Liushen): provided by Henan Tailong Pharmaceutical Co., Ltd.

Blank sample 3 (excluding chicken inner gold, Liushen): provided by Henan Tailong Pharmaceutical Co., Ltd.

Reference substance: chicken internal gold reference medicine (batch number: 121153-201002), ferulic acid reference substance (batch number: 110773-201614), rutin reference substance (batch number: 100080-201610), all provided by China Food and Drug Administration Institute .

Instruments: ZF7C three-use UV analyzer, AL-204 electronic analytical balance, HQ-500DE ultrasonic cleaner; vacuum suction filter, (1cm × 30cm) chromatography column, thin layer color spray device.

1.2 method

(1) Preparation of the test solution: Take 10 pieces of puer compound chicken inner chewable tablets, grind finely, weigh about 4g, add 75% methanol 25ml, ultrasonic for 30min, filter, upper AB-8 macroporous adsorption resin column (The column is 1.8cm, the column height is 18cm, and the column is packed by wet method), washed with water until the water washing liquid is nearly colorless, and then eluted with 60ml of 705% ethanol and 70% ethanol in sequence, and 70% ethanol eluent is collected. , evaporated to dryness, and dissolved in 5 mL of methanol to prepare a test solution.

(2) Preparation of negative sample solution: Take 10 samples of negative sample 1, negative sample 2 and blank sample 3, respectively, and prepare negative sample solution 1, negative sample solution 2 and blank sample solution 3 according to the preparation method of the test solution.

(3) Preparation of reference solution: Take 1 g of the chicken reference drug, and prepare a reference drug solution with the above test solution preparation method.

(4) Take the ferulic acid reference substance and the rutin reference substance, add methanol to make a mixed solution containing 0.5 mg of ferulic acid and 0.5 mg of rutin per 1 ml as a reference solution.

(5) According to the thin layer chromatography test, 5 μl of each of the above six solutions was taken and placed on the same high-efficiency silica gel G thin-layer plate, and the volume ratio of chloroform-ethyl acetate-methanol-water was used (12:3: 8:1), the lower layer solution placed below 10 °C is used as a developing solvent, unrolled, taken out to dry, sprayed with 10% sulfuric acid ethanol solution, heated at 105 ° C until the spots are clear, in sunlight and ultraviolet light (365 nm) View.

1.3 Results and conclusions

The test solution has corresponding spots in the spot of the chicken gold reference drug solution and the reference solution. The negative sample solution 1 had no corresponding spots at the spot of the chicken-gold reference drug solution. The negative sample solution 2 had no corresponding spots at the spots of the ferulic acid and rutin reference solution. The blank sample solution 3 had no corresponding spots in the spot of the chicken internal gold reference drug solution, ferulic acid and rutin reference solution.

The specific thin-layer chromatographic identification conditions of the present invention were used to identify the chicken chewable tablets in the chicken compound, and the identification method had no blank interference, and the components did not interfere with each other, and the specificity was strong and the operability was strong.

2. Determination method of the content of compound chicken inner chewing tablets in children

2.1 Instruments and reagents

Reagents: methanol, acetonitrile, isopropanol for chromatographic purity, ethanol, glacial acetic acid for analytical purity. Neutral alumina, chromatography column.

Instrument: Agilent 1260 high performance liquid chromatograph; Agilent 1290 Infinity ELSD; AL-204 electronic analytical balance; HQ-500DE ultrasonic cleaner; DK-96-II constant temperature water bath; vacuum concentration device, (1cm × 30cm) chromatography Columns, etc.

Sample to be tested - Pediatric compound chicken inner chewing tablets (batch number: 1610222, 1.2g / piece): provided by Henan Tailong Pharmaceutical Co., Ltd.

Negative sample (excluding Liushen): provided by Henan Tailong Pharmaceutical Co., Ltd.

Control substance: artemisinin reference substance (batch number: 100202-201606), p-hydroxybenzaldehyde reference substance (batch number: 201613), Xan Erting reference substance (batch number: 201609).

2.2 Methods and results

(1) Preparation of the test solution: Take the chicken chewable tablets in the compound chicken, grind and smash, accurately weigh 10g, place it in a conical flask, add 25ml of methanol precisely, close the plug, weigh the weight, and sonicize (power 250W, frequency 40kHz) 30 minutes, take out, let cool, then weigh the weight, make up the lost weight with methanol, shake, filter, and accurately measure 10ml of the filtrate, add to the neutral alumina column (100 ～120 mesh, weight 10 g, inner diameter 1 cm), eluted with 50 ml of absolute ethanol, the eluate was collected, concentrated to dryness under reduced pressure at 50 ° C, dissolved in a suitable amount of purified methanol, transferred to a 2 ml volumetric flask, and added. Chromatographically pure methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

(2) Preparation of reference solution: Take appropriate amount of artemisinin, p-hydroxybenzaldehyde and Xan Erting reference substance, accurately weighed, add chromatographic pure methanol to make 0.1mg of artemisinin per 1ml, p-hydroxybenzaldehyde 0.2 Mg, Xanthine 0.1mg mixed reference solution.

(3) Assay method: accurately draw 20 μl of each of the reference solution and the test solution, and inject into a liquid chromatograph, and measure, that is, obtain. Chromatographic conditions: octadecylsilane bonded silica as the stationary phase, methanol-water-glacial acetic acid (85:15:0.45) as mobile phase A, acetonitrile-isopropanol-water (50:20:30) For mobile phase B, gradient elution was carried out according to the following table. The column temperature was 38 ° C, the mobile phase flow rate was 1 ml per minute; the evaporative light scattering detector was used to detect; the theoretical plate number should be no less than 6000 in terms of artemisinin.

Table 1

Time (minutes)	Mobile phase A (%)	Mobile phase B (%)
0	0	100
12	0	100
20	30	70
32	85	15
34	15	85
46	15	85

(4) Results Each tablet contains 0.016 mg of artemisinin, 0.104 mg of p-hydroxybenzaldehyde, and 0.050 mg of Xanthium.

2.3 Determination of methodological methods

2.3.1 Linear relationship test

Take appropriate amount of artemisinin, p-hydroxybenzaldehyde and Xan Erting, accurately weighed, add methanol to prepare mixed reference solution 1-5, artemisinin, p-hydroxybenzaldehyde and Xanthium in each reference solution The respective concentrations of the kiosks are shown in Table 1, respectively, and injected into a liquid chromatograph, and measured according to the chromatographic conditions described in 2.2 step (3).

Linear regression was performed with the concentration of the reference substance as the abscissa (X) and the peak area as the ordinate (Y), and the linear regression equations of artemisinin, p-hydroxybenzaldehyde and Xanthing were obtained respectively.

Results Artemisinin showed a good linear relationship in the range of 1.5ug~24ug. The linear regression equation was Y=5004X-39396. The para-hydroxybenzaldehyde showed a good linear relationship in the range of 1ug~16ug. The linear regression equation is Y=9426.5. x+33214; Xan Erting has a good linear relationship in the range of 0.5ug to 8ug, and the linear regression equation is Y=3118.9x-2778.1. The results are shown in Table 2 and Figure 1, Figure 2, and Figure 3, respectively.

Table 2

2.3.2 Specificity

Preparation of negative sample solution: Take a negative sample (excluding Liushen), and prepare a negative sample solution lacking Liushen according to the preparation method of 2.2. Step (1).

Separately extract the 20 ul of the test solution obtained in step (1), the reference solution obtained in step (2), and the negative test solution in the absence of Liuqu, respectively, into the liquid chromatograph, as described in step 2.2 (3). The chromatographic conditions were determined.

RESULTS: The test solution had the corresponding peak at the same time as the reference solution retention time; the negative test solution lacking Liushen had no corresponding peak at the same retention time as the reference solution. It indicates that the detected components are more prominent, that is, from the six divine songs, the method is specific.

2.3.3 precision

Prepare the guar compound chicken inner chewable tablet for the test solution according to the method described in step (1) of 2.2, and repeat the injection into the liquid chromatograph 6 times, and measure according to the chromatographic conditions described in 2.2 step (3). 3.

Table 3 precision results

From the above results, it is understood that the method for testing the content of the pediatric compound chicken inner chewable tablet of the present invention has good precision.

2.3.4 Stability of the sample solution to be tested

Prepare the test solution according to the preparation method of step 2.2 (1), and place the test solution for 0, 2, 4, 6, 8, 12, 24 h at room temperature, respectively, at the above time point, according to step 2.2 ( 3) The chromatographic conditions determine the content of artemisinin, p-hydroxybenzaldehyde, and chlorhexidine in the sample, and calculate the RSD value. The results are shown in Table 4.

Table 4 Determination of the stability of sample solution in chicken compound chicken chewable tablets

The results show that the test solution prepared by the method of the present invention has good stability within 24 hours at room temperature.

2.3.5 Reproducibility test

Take six parts of 161022 batches of pediatric compound chicken inner chewable tablets, prepare the test solution according to the method of 2.2 step (1), and determine the content according to the chromatographic conditions described in 2.2 step (3). The results are shown in Table 5.

Table 5 Reproducibility test for the determination of the content of chicken chewable tablets in children

The results show that the content determination method of the present invention has good reproducibility.

2.3.6 Recovery test

Six samples of pediatric compound chicken inner chewable tablets with known content (artemisinin 0.151 mg/tablet, p-hydroxybenzaldehyde 0.093 mg/tablet, Xan Erting 0.0487 mg/tablet) were accurately weighed according to 2.2 steps ( The method of 1) is made into six test solutions. Accurately weigh the reference substance of artemisinin, the reference substance of p-hydroxybenzaldehyde and the reference substance of Cangting, and make a mixture of 0.2521mg of artemisinin, 0.1559mg of p-hydroxybenzaldehyde and 0.0813mg of Xanthing Pavilion per 1ml. 100 ml of the solution was added to each of the six samples to 2 ml of the control. The content was determined according to the chromatographic conditions described in step 2.2 (3), and the recovery rate was calculated according to the Chinese Pharmacopoeia 2015 edition of the four parts of the 9101 drug quality standard analysis method verification guidelines. The results are shown in Tables 6, 7, and 8.

Calculation formula:

Table 6 Artemisinin sample recovery rate results

Table 7 results of the recovery of p-hydroxybenzaldehyde

Table 8 Cangting Pavilion sample recovery rate results

In summary, the method for determining the content of the compound chicken inner chewable tablet of the invention is strong in specificity, high in precision and good in reproducibility, and can effectively control product quality.

Comparative example 1-6

The pediatric compound chicken inner chewable tablets were identified according to the method described in 1.2 of Example 1, except that the developing agent used in Example 1 was replaced with the following developing agent:

a. ethyl acetate-formic acid-water (8:1:1)

b. chloroform-acetone (95:5)

c. n-Butanol-glacial acetic acid-water (7:1:1)

d. n-butanol-glacial acetic acid-water (8:2:1)

e. n-Butanol-Ethanol-glacial acetic acid-water (4:1:1:2)

f. chloroform-ethyl acetate-methanol-formic acid (40:5:10:0.2)

As a result, there was a negative interference under the condition of the b-developing agent, and the spots could not be completely and effectively separated under the conditions of a, c, d, e, and f developing agents.

Comparative Example 7-10

The content of pediatric compound chicken inner chewable tablets was determined according to the method described in 2.2 of Example 1, except that the mobile phase and elution conditions used in Example 1 were replaced by the following mobile phases and elution conditions:

a. Using octadecylsilane-bonded silica gel as a filler, methanol-water-glacial acetic acid (85:15:0.45) as mobile phase A, n-hexane-isopropanol-water (20:50:30) For mobile phase B, perform a gradient elution as specified in the table below.

Time (minutes)	Mobile phase A (%)	Mobile phase B (%)
0	0	100
10	0	100
60	100	0

b. Using octadecylsilane-bonded silica gel as a filler, methanol as mobile phase A, and 0.4% phosphoric acid as mobile phase B, gradient elution as specified in the table below.

Time (minutes)	Mobile phase A (%)	Mobile phase B (%)
0	100	0
10	80	20
60	55	45

c. Using octadecylsilane-bonded silica gel as a filler, acetonitrile: 0.2% acetic acid (60:40) as a mobile phase.

d. Using octadecylsilane-bonded silica gel as a filler, methanol-water-glacial acetic acid (85:15:0.45) as mobile phase A, and acetonitrile-isopropanol-water (50:20:30) as Mobile phase B, gradient elution as specified in the table below.

Time (minutes)	Mobile phase A (%)	Mobile phase B (%)
0	0	100
10	0	100
60	100	0

In the case of the above ac mobile phase, the reference solution and the test solution were respectively tested. As a result, both methods b and c have unexpressed main peaks, and the retention time of method a is relatively late, the mobile phase span gradient is large, and the peak Poor shape. In the case of method d, the main peak of each component is obvious, but the peak shape is poor.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solutions of the present invention within the scope of the technical idea of the present invention. These simple variants All fall within the scope of protection of the present invention.

Claims (11)

1. A method for determining and contenting a compound chicken inner chewing tablet, characterized in that the method comprises:

   (1) a step of identifying by thin layer chromatography, wherein

   The conditions of the thin layer chromatography are as follows: a thin layer chromatography plate of silica gel G is used as a thin layer chromatography plate, and the lower layer is placed under chloroform-ethyl acetate-methanol-water (12:3:8:1) at 10 ° C or lower. Liquid is a developing agent;

   (2) a step of performing content determination by high performance liquid chromatography, wherein

   The high performance liquid chromatography conditions are as follows: octadecylsilane bonded silica as a stationary phase, methanol-water-glacial acetic acid in a volume ratio (85:15:0.45) as mobile phase A, and acetonitrile-isopropyl The alcohol-water was mobile phase B in a volume ratio (50:20:30), and the gradient elution was carried out according to the following conditions:

   Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 0 100 12 0 100 20 30 70 32 85 15 34 15 85 46 15 85
2. The method for identifying and contenting a pediatric compound chicken inner chewable tablet according to claim 1, wherein the step of identifying according to step (1) further comprises using a macroporous adsorption resin column for the pediatric compound chicken inner gold chewable tablet. The step of chromatography.
3. The method for identifying and contenting a pediatric compound chicken inner chewable tablet according to claim 2, wherein the step of macroporous adsorption resin column chromatography comprises: cultivating the compound chicken inner gold chewable tablet with a mass fraction of 75% methanol solution is mixed at a weight-to-volume ratio of 4:25, sonicated, and then subjected to macroporous adsorption resin column, preferably using AB-8 macroporous adsorption resin column for chromatography. After elution adsorption equilibrium, the mass fraction is 70%. The ethanol solution is used as an eluent to elute the macroporous adsorption resin column, and the eluate is collected, evaporated to dryness, and an analytically pure methanol solution is added.
4. The method for identifying and contenting a pediatric compound chicken inner chewable tablet according to any one of claims 1 to 3, wherein the step of identifying the step (1) uses a chicken gold-methanol solution as a reference drug solution. A ferulic acid-rutin-methanol solution was used as a reference solution.
5. The method for identifying and contenting a pediatric compound chicken inner chewable tablet according to any one of claims 1 to 4, wherein the step of identifying the thin layer chromatography in the step (1) comprises:

   (1) preparing a sample solution for the pediatric compound chicken inner gold chewable tablet according to the step of macroporous adsorption resin column chromatography;

   (2) preparing a chicken gold-methanol reference drug solution according to the step of macroporous adsorption resin column chromatography on the chicken internal gold reference material;

   (3) taking ferulic acid reference substance and rutin reference substance, adding methanol to obtain ferulic acid-rutin-methanol reference solution;

   (4) The above three solutions were aspirated and identified according to the conditions of the thin layer chromatography.
6. The method for determining and contenting a pediatric compound chicken inner chewable tablet according to claim 5, wherein the step of identifying the thin layer chromatography in the step (1) comprises:

   (1) The pediatric compound chicken inner chewable tablets were pulverized, mixed with a mass fraction of 75% methanol solution at a weight-to-volume ratio of 4:25, sonicated for 30 min, filtered, and the upper AB-8 macroporous adsorption resin column layer Analyze, wash with water until the washing liquid is nearly colorless, then elute with 35% ethanol and 70% ethanol in sequence, collect 70% ethanol eluent, evaporate dry, add methanol to dissolve, then get the test solution ;

   (2) taking chicken internal gold reference medicine, according to the method of step (1) to obtain a chicken gold-methanol reference medicine solution;

   (3) taking ferulic acid reference substance and rutin reference substance, adding methanol to make a mixed solution containing 0.5 mg of ferulic acid and 0.5 mg of rutin per 1 ml as a reference solution;

   (4) Aspirate the above three solutions, respectively, on a high-performance silica gel G thin-layer plate, and use chloroform-ethyl acetate-methanol-water to develop a volume ratio of 12:3:8:1 as a developing agent, and take it out to dry. Spray with a 10% by mass solution of sulfuric acid in ethanol, heat at 105 ° C until the spots are clear, and examine them under sunlight and ultraviolet light (365 nm).
7. The method for identifying and measuring a pediatric compound chicken inner chewable tablet according to any one of claims 1 to 6, wherein the content determining step according to the step (2) further comprises: using the pediatric compound chicken inner gold chewable tablet The step of purifying the neutral alumina column.
8. The method for identifying and contenting a pediatric compound chicken inner chewable tablet according to claim 7, wherein the step of purifying the neutral alumina column comprises: pediatric compound chicken inner gold chewable tablet and chromatographic pure methanol According to the weight-volume ratio of 1:2.5, sonicated, purified by neutral alumina column, eluted with neutral ethanol column with absolute ethanol solution as eluent, and the eluent was evaporated to dryness. Methanol.
9. The method for determining and contenting a pediatric compound chicken inner chewable tablet according to any one of claims 1-8, wherein the content determining step according to step (2) is artemisinin-p-hydroxybenzaldehyde- Xanthing - methanol solution is the reference solution.
10. The method for determining the content and content of a pediatric compound chicken inner chewable tablet according to any one of claims 1 to 9, wherein the step of determining the content by the high performance liquid chromatography in the step (2) comprises:

    (1) preparing a test solution by purifying the pediatric compound chicken inner gold chewable tablet according to the neutral alumina column;

    (2) taking artemisinin, p-hydroxybenzaldehyde and Xan Erting reference substance, adding methanol to make a mixed solution of artemisinin-p-hydroxybenzaldehyde-Cangling-methanol as a reference solution;

    (3) Aspirating the above two solutions, injecting into a high performance liquid chromatograph, and performing content determination according to the high performance liquid chromatography conditions.
11. The method for determining and contenting a pediatric compound chicken inner chewable tablet according to any one of claims 1 to 10, wherein the step of determining the content by the high performance liquid chromatography in the step (2) comprises:

    (1) The pediatric compound chicken inner chewable tablets were pulverized, mixed with the chromatographic pure methanol solution at a weight-to-volume ratio of 1:2.5, sonicated for 30 minutes, allowed to cool, filtered, and passed through a neutral alumina column (100~). Purification by 120 mesh), eluting with absolute ethanol, collecting the eluent, concentrating under reduced pressure, dissolving in chromatographically pure methanol, and filtering to obtain a test solution;

    (2) Take artemisinin, p-hydroxybenzaldehyde and Xan Erting reference substance, add methanol to make a mixed solution containing 1mg of artemisinin, 0.2mg of p-hydroxybenzaldehyde and 0.1mg of Xanthing. Solution

    (3) Aspirating the above two solutions and injecting them into a high performance liquid chromatograph, the high performance liquid chromatography conditions are: using octadecylsilane bonded silica as a stationary phase, the column temperature of the column is 38 ° C, with methanol - Water-glacial acetic acid was mobile phase A in a volume ratio (85:15:0.45), and mobile phase B in a volume ratio (50:20:30) in acetonitrile-isopropanol-water. Gradient elution was carried out according to the following conditions. The flow rate of the mobile phase is 1 ml / min;

    

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